The present invention relates to drug screening methods and methods of preventing neural tissue damage caused by xcex1-synuclein aggregation. These methods are especially useful in the design and development of inhibitors of Lewy body diseases and other synucleinopathies, and further useful in the treatment of such neurodegenerative diseases, particularly Parkinson""s Disease.
Parkinson""s disease (PD) is a common neurodegenerative disorder and was first described by James Parkinson in 1817. The four primary diagnostic signs of the illness are resting tremor, bradykinesia, muscular rigidity and postural instability. These signs of motor deficiency result from the loss of dopaminergic neurons in the nigrostriatal system [Gibb, W., et al., J. Neurol. Neurosurg. and Psych., 51:745-52 (1988)].
PD is characterized by the formation of Lewy bodies and death of dopaminergic neurons. [Adams D. et al., Principles of Neurology, 874-880, 3rd Edition, McGraw-Hill, N.Y., (1985)]. The neuropathological hallmark of PD is the Lewy body. Lewy bodies are intracytoplasmic inclusions that occur in degenerating neurons which are composed of a dense core of filamentous and granular material surrounded by radically oriented filaments that have a diameter of 10-20 nm [Goedert, 20 M., et al., Curr. Op. Neurobio. 8:619-32 (1999)]. In general, the causes of PD are not known and there has been vigorous debate over the relative roles of genetics and environmental factors [Tanner, C., et al., JAMA, 281:341-6 (1999)]. Exposure to manganese precipitated a Parkinsonian syndrome in miners which also includes schizophrenia form behaviors. Some epidemiological studies have found an association between industrial exposure to iron and the incidence of PD [Corell J. M et al., Toxicol. Appl. Pharmacol., 80:467-72, (1985)], between incidence of PD and blood mercury levels [Ngim C. H. et al., Neuroepi., 8(3):128-141 (1989)] and with death rates from PD and proximity to iron related industrial processed [Rybicki A. et al., Mov Disord., .8(1):87-92_(1993)].
xcex1-Synuclein was originally identified as a protein that is upregulated associated with neuron outgrowth during the critical period of Zebra finch song learning [George M., et al., Neuron, 15:361 (1995)]. xcex1-Synuclein is a ubiquitous protein that shares significant physical and functional homology to the protein chaperone, 14-3xe2x80x943, and is particularly abundant in the brain (Ostrerova N. et al., J. Neurosci., 19:5782 (1990); Clayton D. et al., TINS 21:249 (1998)]. xcex1-Synuclein is normally phosphorylated at serines 87 and 129. (Okochi M. et al., J. Biol. Chem., 275:390 (2000)]. Recent studies showed that mutations in xcex1-synuclein can cause familial PD and that xcex1-synuclein accumulates in Lewy bodies. These discoveries suggest that xcex1-synuclein participates in the pathophysiology of PD. (Spillantini M. et al., Nature, 388:839 (1997); Spillantini M. et al., PNAS USA, 95:6469 (1998); Jenner P. et al., Ann. Neurol., 44:S72 (1998)]. The only identified mutations associated with familial PD to date are the A53T and A30P mutations in the xcex1-synuclein gene (Goedert, M., et al., Curr. Op. Neurobio., 8:619-32 (1999); Papadimitriou, A., et al., Neurology, 52:651-4 (1999); Polymeropoulos, M., et al., Science, 276:1197-9 (1997)]. However, there has been much circumstantial evidence implicating oxidative stress in the etiology of the disease (Jenner, P., et al., Annual Neurol., 44:S72-84 (1998)].
A variety of experimental evidence suggests that Lewy bodies interact with xcex1-synuclein. For example, immunohistochemical studies indicate that Lewy bodies stain strongly for xcex1-synuclein and ubiquitin (Jenner, P., et al., Annual Neurol., 44:S72-84_(1998); Markopoulou, K., et al., Annual. Neurol., 46:374-81 (1999); Spillantini, M., et al., Nature, 388:839-40 (1997); and Spillantini, M, et al., Proc. Natl. Acad. Sci. USA, 95:6469-73 (1998)]. In vitro experiments using recombinant protein suggest that the mutations, A53T and A30P, increase xcex1-synuclein aggregation in comparison with the wild type xcex1-synuclein (Conway, K., et al., Nature Med., 4:1318-20 (1998); Giasson, B., et al., J. Biol. Chem., 274:7619-22 (1999); Hashimoto, M., et al., Brain Res., 25 799:301-6 (1998)].
One of the important questions regarding xcex1-synuclein aggregation and Lewy body formation is whether these processes harm the cell. Lewy bodies could either be inert tombstone markers that occur in response to free radical damage, or they might be toxic agents that harm the cell. Examples of both situations exist in the literature. Aggregated amyloid-xcex2 (Axcex2) is toxic to neurons, while lipofuscin appears to be innocuous to cells (Behl, C., et al., Cell 77:817-27 (1994)]. The Huntington""s protein presents an intermediate situation where the toxicity associated with Huntington""s appears to precede aggregation, and aggregation of Huntington""s protein might even be protective [Saudou, F., et al., Cell 95:55-66 (1998)]. Our own previous studies showed that transient over-expression of xcex1-synuclein is toxic to a variety of cells, including two neuronal cell lines, SK-N-SH and PC12 [Ostrerova, N., et al., Neurosci., 19:5782-91 (1999)]. Consistent with this observation, Masliah and colleagues have recently shown that mice over-expressing xcex1-synuclein show an age-related loss of dopaminergic terminals and motor impairment, which could be indicative of toxicity [Masliah, E. et al., Science, 287:1265-1269 (2000)]. These findings suggest that an increased rate of xcex1-synuclein aggregation might contribute to the mechanisms of neurodegeneration in PD and other Lewy body diseases.
Recent studies on transgenic animals also suggest that aggregation of xcex1-synuclein is harmful to neurons. It was recently reported that dopaminergic dysfunction occurred in transgenic mice expressing wild type human xcex1-synuclein [Masliah, E., et at., Science, 287:1265-1269 (2000)]. Further, it was reported that Drosophila over-expressing xcex1-synuclein exhibited dopaminergic dysfunction and dopaminergic neuronal death associated with development of xcex1-synuclein aggregates [Feany, M B, et al., Nature 404:394-8 (2000)]. Evidence suggests that neurons with dopamine develop xcex1-synuclein aggregates and degenerate as these aggregates development.
Recently, oxidative stress produced by iron and hydrogen peroxide has been shown to induce amyloid-like aggregate formation of xcex1-synuclein in vitro [Hashimoto, M., et at., NeuroReport, 10:717-21 (1999); Paik, S., et al., Biochem. J., 340:821-8 (1999)]. Oxidative stress is thought to contribute to PD because dopamine, which is a strong free radical generator, is the principle neurotransmitter in the substantia nigra [Chiueh, C., et at., Adv. Neurol., 60:251-8 (1993); Jenner, P. et al., Ann. Neurol., 25 44:S72-84 (1998)]. In addition, iron, which also stimulates free radical production, accumulates in the substantia nigra with age [Jenner, P., et al., Ann. Neurol., 44:S72-84 (1998)]. Iron is deposited as hemosiderin granules in the cytoplasm, and mitochondria filled with ferritin granules have been observed in the neuronal and glial cells of the ventorlateral thalamus, caudate and lenticular nuclei and substantia nigra of Parkinsonian brains. [Earle M., J. Neuropathol. Exper. Neurol., 27(1):1-14, (1968); Asenjo A. et al., Rev. Neurologique, 121 (6):581-92, (1969); Riederer P., et al., J. Neurochem., 52(2):515-20, (1989)]. Thus, the oxidative conditions present in the substantia nigra could promote xcex1-synuclein aggregation. However, in the prior art, whether such oxidative conditions actually promote a-synuclein aggregation in living neurons is unknown.
A need, therefore, exists for substances that will inhibit Lewy body formation, which can be used to treat diseases such as PD. At the same time, ways to find such substances, through screening assays, are also needed in the art. U.S. Pat. No. 6,184,351, discloses that synuclein aggregation can be induced by continually shaking xcex1-synuclein for long periods of time at very high concentrations. However, the required high concentration and continuous shaking is neither physiologic nor conducive to the development of screening assays. Moreover, the assay described in the ""351 patent does not directly measure xcex1-synuclein aggregation; rather the soluble, unaggregated protein is determined. Further, the ""351 assay is directed only to detecting nucleation-affecting agents, and not agents that would inhibit aggregation by other mechanisms. Therefore, the art would benefit from better methods for screening drugs useful for the inhibition of Lewy body formation.
The present invention relates to methods of identifying factors that lead to the inhibition of the aggregation of xcex1-synuclein in vitro or in living neurons.
It is an objective of the present invention to provide a method of identifying, screening, and modeling pharmaceutical agents capable of inhibiting the aggregation pathway of xcex1-synuclein. The pharmaceutical agents are, for instance, cations, small molecules, peptides, peptide mimetic compounds, nucleic acids, complex sugars, such as heparin analogues, or combinations thereof.
It is also an objective of the present invention to design, make, and use anti-xcex1-synuclein aggregation drugs for treating a neurodegenerative condition, such as PD, Alzheimer""s disease, diffuse Lewy body disease, mixed AD-PD, multiple system atrophy and Hallervorden-Spatz disease.